The innate immune system is of fundamental importance in protection against infectious diseases. Macrophages are among the "first responders" to a microbial challenge, acting as sentinels against infectious agents. Their response to this challenge is rapid, requiring a coordinated signal transduction apparatus to trigger production of pro-inflammatory cytokines, such as TNF. Using a proteomics approach to phagocytic signaling, we identified B-cell adaptor protein (BCAP) as undergoing enhanced tyrosine phosphorylation during phagocytosis in macrophages, BCAP was previously implicated in the PI 3-kinase and NF-B pathways in B-cells. Unexpectedly, we found that macrophages derived from BCAP-/- mice displayed profound defects in innate immune signaling, such as LPS-stimulated TNF release. We hypothesize that BCAP represents a previously unrecognized component of the innate immune apparatus of macrophages. In this proposal, we outline two Specific Aims: We will determine the mechanism by which BCAP contributes to TLR signaling in mouse macrophages and determine the spectrum of innate immune receptors that require BCAP for their function. We will delineate specific signaling pathways (e.g., NF-B, ERK, JNK, p38 MAP kinases) that require BCAP as an intermediary. We will test the hypothesis that BCAP serves as an adaptor for PI 3-kinase in signaling via TLR4. We will test the novel hypothesis that BCAP acts as a "counterbalance" to the inositol phosphatase, SHIP, thus "setting the gain" for PI 3-kinase-dependent signaling. Because the defect in LPS signaling is most marked in cells co-incubated with IFN, we will determine whether BCAP has an independent role in IFN-? signaling. In Specific Aim 2, we will determine whether BCAP plays a role in the innate immune response to endotoxin in an experimental model of aseptic peritonitis. Because our goal is to study the role of BCAP in macrophage, rather than in B-cells, we will cross BCAP"'' mice with congenic MT B1-deficient mice to obtain mice with BCAP-deficient macrophages but lacking B1 cells. We will test these mice for responses to an intraperitoneal challenge of endotoxin. We will determine survival, peritoneal exudate cellular and cytokine composition. We will test the hypothesis that BCAP is required for optimal production of pro-inflammatory cytokines. Collectively, these in vitro and in vivo studies will establish the role that BCAP plays in innate immunity in macrophages. These studies are of fundamental importance to public health. They are applicable to understanding the role of white blood cells in a multitude of infectious diseases, including agents of biological warfare. [unreadable] [unreadable] [unreadable]